I am trying to clone a 12kb insert into a pBS vector (~4kb) using In-Fusion cloning which uses 15bp homologous ends instead of TA cloning. I linearized the vector with EcoRV which is a blunt end digestion enzyme and gel purify it. I am also using Takara's LAPCR to amplify the 11kb insert and gel purify it as well. I go through the ligation, trans, and plating, however when I screen the colonies I get some very weird results.
First, using digestion enzymes to linearize the vector and vizualize it on a gel, I only get a length of around 7-8 kb which would mean that my insert is only ~3-4kb instead of 12kb. I"m not sure why this is happening, or how this would even happen. Also, to confuse things even more, I have been screening these inserts by doing a basic PCR using primers that target the 3', 5' and middle section of the insert and they all amplify at the correct lengths which would suggest that the entire insert is present in the vector. But the digestion says otherwise.
Edited by Zach T, 09 June 2011 - 02:49 PM.